Protease Nexin (PN) is a newly identified cell-secreted protein that binds and inhibits the serine proteases thrombin, urokinase, plasmin, trypsin and elastase. PN transports bound proteases to cell surface receptors, that mediate the endocytosis and degradation of the proteases. Because a wide variety of cultured mammalian cells secrete PN, we have recently purified the human form of this inhibitor and now propose to define its biochemical nature and physiological functions. 1) We will obtain a partial amino acid sequence of purified human fibroblast PN in order to establish whether PN belongs to the family of protease inhibitors that includes antithrombin III and alpha-1-proteinase inhibitor. Sequencing will also provide information about the protease specificity of this inhibitor by revealing the residues at its reactive center. 2) We will determine the rate constants for the association of PN with a variety of serine proteases. 3) Using serum-free cell cultures the possible role of PN in regulating cell-secreted plasminogen activators and other serine proteases will be investigated with cultured normal and malignant human cells. The experiments with malignant cells are pertinent because cancerous cells frequently exhibit uncontrolled secretion of serine proteases. We will investigate whether addition of anti-PN antibody to cell cultures results in increased protease activity in the culture medium, and alters cell morphology, movement or growth. An attempt will be made to detect and identify protease-PN complexes that form in cell culture medium. 4) Finally, normal and malignant human tissues will be screened for active PN and PN antigen. Localization of PN in tissue sections will be determined at the light microscope level using immunochemistry and standard histological techniques.